1. Filed of the Invention
The present invention relates to the measurement of luminescence by a luminescence microplate reader. In particular, the present invention relates to a luminescence validation microplate which is used to validate the reliability of the information received from a luminescence assay microplate during its reading in a luminescence microplate reader.
2. Description of Related Art
The growth of biological research, reporter gene assays and new pharmaceutical compound screening and in some cases medical diagnostics has created a need for handling large numbers of test samples at one time in order to control costs and efficiently handle these large numbers of samples. A number of analytical methods are now available for high throughput screening of these samples. One of those important methods which are in increasing use is the use of luminescence. It has been discovered that various substrates both naturally occurring in nature and man-made have luminescence properties at a given wavelength and can be used as a tag or marker for various test assays where luminescence can be used as a direct measure of some other activity movement or the like. Often called luminophors these compositions are readily available in various wavelengths intensities and utilities. Techniques for using with these substrates are similar to fluorescence emission and radioactive measurement techniques. Typically, large numbers of samples are processed for luminescence emission in a multi-well sample plate called an assay microplate. These microplates are typically rectangular and provide an array of wells, usually 24 or 96 wells in typical examples, but 384 well and 1536 well microplates are becoming more common as well. Most common plates are 96 well (in 8 by 12 configuration) at this time and all plates regardless of the number of wells, comprise roughly the same rectangular size. While originally variations that were significant existed between manufacturers of these plates requiring plates matching the reader, new standards promulgated by SBS/NIST now exist standardizing not only the rectangular size of the plates but the position and size of the wells for the various multi-well configurations. New standards allow for manufacture of microplates which fit all manufacturers' machines and allow for a reduction in problems associated with their use. Notable, as well numbers per plate increase, well diameter decreases and these standardizations have become even more critical.
Assay microplate wells are filled with test samples and then placed in a detector system of the luminescence microplate reader, for measuring the relative luminescence emissions of each test well. Since different luminescence materials use for microplates assays produce different degrees of light intensity. Light intensity can a direct measure of test results, i.e. the greater the light intensity the greater the result. Detectors therefore usually need to be capable of detecting a wide range of light intensities.
Although luminescence microplates and luminescence microplate readers are of great utility in automated screening, there are a number of critical issues concerned with their use that affect the reliability of their use. A luminescence microplate reader has a series of optical devices wherein each device is positioned to correspond to a well in the microplate that holds a test sample. The optical device is often a photomultiplier tube. In the alternative, the luminescence microplate reader is fitted with a single optical device and the plate, the reading device or both are moved to the appropriate reading position. Use of the microplate on the luminescence microplate reader must involve alignment of each sample well with the optical device of the luminescence microplate reader. Movement of the microplates or optical devices is usually done using stepper motors wherein the movement is guided on a certain number of (factory calibrated) steps from a “home” position. Alignment can be adversely affected when one or more of the aligned components involved is shifted in position or becomes damaged. If, for any reason, the alignment is incorrect, the wells will not be centered properly in alignment with the optical reading device, resulting in an incorrect luminescence measurement. IN addition to optical problems the plate carrier can be physically bent or otherwise misaligned with the original adjustment parameters of the reader which also produces a bad measurement.
The optics used to measure luminescence must avoid detecting transmission of luminescence from one sample well as the luminescence from an adjacent well. This adjacent well detection problem is an alignment problem called “cross-talk”. Cross-talk is extremely undesirable because it means the emissions detected, by a particular optical device at a given location originated from the test sample of a different well. In a worse case scenario, a particular well optical device is detecting luminescence cross-talk for all the surrounding or adjacent wells. Depending on where the well is located this can be up to 8 adjacent wells in a standardized microplate set-up. Even in a best worse-case scenario cross talk from a single adjacent well is extremely undesirable.
Linearity is also an important measurement to look at to validate the accuracy of the readings of the luminescence microplate reader. Linearity is a measure of the relationship between different amounts of luminescence emissions in a series of wells as measured by their light output and is usually measured from darkness or close to darkness to saturation of the detector ability to sense light. A linear response relationship should exist between the measured luminescence and the strength of the luminescence emissions. Linearity is an indication of the relative concentration of luminescence emissions in the series of wells. As the optical, electronic and other components of the luminescence microplate reader age, the detection efficiency can be diminished to a lesser or greater degree for some measurements versus others and this seems to be especially the case at the high and low ends of emission measurements. To some extent these problems can be addressed by software programs and by advancing technologies which slow the aging process of the readers. However, these solutions do not solve all the linearity problems with luminescence microplate readers and the need to calibrate the accuracy of the machines remain.
A number of luminescence validity microplates have been developed to accommodate one or more of these problems. For Example in U.S. Pat. No. 6,335,997, there is disclosed a device for testing luminescence microplate readers. It disclosed a means for delivering a series of low level light sources ranging over several orders of magnitude. This is accomplished by shining a light source on a series of fiber optic collection rods where the light for each is attenuated via an opaque sleeve on each rod. Light is delivered to the reader via an output aperture connected to the chamber through drilled tunnels. This construction delivers a means for producing a linearity test. However, the size of the rods and their placement leaves no room for any other type of validity testing on a validity plate of standard size. Further, opaque sleeves can move and easily become unadjusted and change the output of the light conducted to the light output aperture. Another problem is since the fiber optic rods are not directly connected to the light source, the amount of light reaching the far rods can seriously decrease compared with the near in rods, due to both the distance traveled and the interposing other rods. Other luminescence validity microplates are available but all currently suffer from similar problems.